In mammalian cells, TCF (Ternary Complex Factor) transcription factors belongs to the ets 1 family. They share through their ets DNA-binding domain (85 amino acids) the ability to bind to the Serum Responsive Element (SRE) when associated to SRF dimers (Serum Response factor). The ternary complex formation requires the presence of a SRE consensus sequence SEQ ID No: 17 (C/A)(C/A)GGA(A/T) next to a SRF consensus site SEQ ID No: 16 (CC(A/T)6GG) without any orientation requirement. The neighbouring of these two sequences in several immediate-early gene promoters induces their transcription in response to mitogens as serum or growth factors but also in response to phorbol esters and stress stimuli. These sequences were first identified in the c-fos promoter on which Ets proteins and SRF are constitutively bound.
Known members of the TCF family members are Elk-1, SAP-1 and Net (SAP-2, ERP). These proteins are widely expressed.
This sub-family of TCF transcription factors is defined by a strong sequence homology pattern that relates to the presence of different functional domains. Starting from amino to carboxy terminal, the members of the TCF family display at least four homology boxes in their polypeptidic sequence:                the A-box also called the ETS DNA-binding domain that is directly involved in binding with the SRE consensus sequence,        the B-box (a short hydrophilic region) is involved in interaction with SRF,        the D-box located upstream to the C-box consists in a MAPK targetting domain,        the C-box and C-terminal sequences contain the phosphorylation sites (six to seven (S/T)P motifs) by MAPKinases (ERKs, JNKs and p38s) and consists in a transcriptional activation domain. The induction of transcriptional activity by TCF requires phosphorylation of the TCF by MAPKs in this regulatory domain.        
The position of A, B, C and D boxes with regards to amino acid sequence of human or murine polypeptide is as follows: A (human)=1-90, B (human)=133-151 , C (human)=321-378, D (human)=290-299 ; A (murine)=1-90 , B (murine)=133-151 , C (murine)=323-380, D (murine)=291-301.
The formation of the ternary TCF-SRF-SRE complex requires at the same time protein/DNA binding (TCF to SRE and SRF to SRF consensus sequence) and the protein/protein interaction between the TCF B-box and SRF. This complex assembly is facilitated by TCF phosphorylation by MAPKs.
Murine Net was cloned in 1994 by Giovane et al. (Genes Dev., 8, 1502-1513 (1994)) and was also reported by T. Liberman et al. (ERP a new member of the ets transcription factor/oncoprotein family: cloning, characterization and differential expression during B-lymphocyte development. Mol. Cell Biol. 1994, 14: 3292-309) (ERP)( Accession number L19953). The human sequence is referred as Z36715 and the murine sequence is referred as Z32815 in GENBANK® (sequence database). Chromosomal localization of murine net gene was mapped to 10C-D1 and its human counterpart on 12q22-23. This last locus is now called ELK3. This localization corresponds to the sap2 gene. The Jackson laboratory and HGMW approved that elk3 is now the common loci for net/erp (murine) and its human match sap-2 (human) loci (Giovane et al. Locations of the est subfamily members of net, elk 1, and sap1 (ELK3, ELK1, and ELK4) on three homologous regions of the mouse and human genomes. Genomics (1995) 29:769-72).
Net in a normal cell context displays repressor activity on transcription. This can be interpreted through a competition for both MAPK phosphorylation and TCF formation. Net antisense stimulates SRE activity when stimulated by serum. The hypothesis of competition at phosphorylation site is validated by the negligible DNA-binding activity of Net protein at the c-fos SRE. A repressive activity on TCF-dependent transcription was also recently described for bHLH transcription factors through a competitive mechanism involving their ETS-domain binding Yates et al. (Id helix-loop-helix proteins inhibit nucleoprotein complex formation by the TCF ETS-domain transcription factors. EMBO J. (1999) 18: 968-76.)
Amongst the TCFs, Net has the particularity to possess also a NID Novel Inhibitory Domain located between aa 153-208, which contains a Helix-Loop-Helix protein-protein interaction motif. This NID inhibits specific DNA binding by Net but also transactivation in the absence of an activated ras signaling (Maira et al. Net (ERP/SAP2) one of the Ras inductible TCFs, has a novel inhibitory domain with resemblance to the helix-loop-helix motif. EMBO J. (1996) 15: 5849-65). Net has also a second inhibitory domain , the CID, aa 274-282, that interacts with CtBP (Criqui-Filipe et al. Net, a negative Ras switchable TCF, contains a second inhibitory domain, the CID, that mediates repression through interactions with CtBP and de-acetylation. EMBO J. (1999) 18 :3392-3403).
The Net C domain integrates the signalling from a range of activators of the MAPK cascades at different MAPK consensus phosphorylation sites (T/S-P). Depending on the nature of this activator, the transcriptional potential of Net is regulated by its subcellular translocation. Net sequence contains a nuclear localization signal (NLS) in the D box and a nuclear export signal (NES) in the Ets DNA binding domain. C. Ducret et al. (the Net repressor is regulated by nuclear export in response to anisomycin, UV and heat shocks M.C.B, 19: 7076-7087) have shown that the JNK pathway induces an active nuclear export of the Net protein. This effect is inhibited by leptomycin B that inhibits the NES binding to CRM1, required for this active nuclear export. The presence of this active NES is specific for Net sequence. Thus, JNKs are involved in Net phosphorylation but this activation doesn't result in transcriptional activation due to this cytoplasmic shuffling. JNK induces nuclear-cytoplasmic shuffling by phosphorylation of the JEX box (aa 233-253). In contrast, transcription activation involves phosphorylation of the C box by p38 and ERK1-2 (Ras) pathway (Ducret et al. Oncogene, in press).
Net transcriptional activity can be activated by oncogenic Ras, Src and Mos but not by an oncogenic Raf, thus showing that ERKs should not be directly involved in Net transactivation.
There is a need in the art to better understand the molecular mechanisms of NET in cellular processes. In particular, there is a need in the art to identify additional NET functions and/or interaction with cellular components. The present invention addresses this need, as discussed below.
The citation of any reference herein should not be construed as an admission that such reference is available as “Prior Art” to the instant application.